This invention relates to a method for the improvement of implantation rates after in vitro fertilization (IVF), for the treatment of infertility and for the treatment and prevention of early pregnancy loss in women with a nitric oxide synthase substrate (L-arginine), a nitric oxide donor or both, alone or in combination with progesterone and/or estrogen.
Human in vitro fertilization is surprisingly unsuccessful. The overall birth rate per IVF treatment cycle is approximately 14% in USA (Medical Research International Society for Assisted Reproductive Technology [SART], The American Fertility Society [1992]. Fertil Steril 5:15 ), and 12.5% in UK (The Human Fertilization and Embryology Authority. Annual Report, London 1992).
Success is greater when more than one embryo is transferred simultaneously. However, simultaneous transfer of multiple embryos increases the incidence of multiple pregnancy and the possibility of miscarriage and prematurity. The reasons for the low pregnancy rates after IVF are still not completely understood. The quality of both the embryo and the uterine environment affects success. Generally, there is a high rate of spontaneous early abortion in fertile cycles in women. After natural conception, possibly as many as 50-60% of very early pregnancies are lost (Winston M L, Handyside A H [1993], New challenges in human in vitro fertilization. Science 260:932-935). This may be due to both conceptus abnormalities and dysynchrony between embryo and endometrium at the time of embryo transfer.
Most losses may be due to abnormalities of the conceptus or the still inappropriate culture conditions, since the success of embryo transfer after IVF decreases as the time after insemination increases (Winston M L, Handyside A H [1993], New challenges in human in vitro fertilization. Science 260:932-935).
To overcome possible deficiencies in culture media, transfer of oocytes (gamete intrafallopian transfer--GIFT) or zygotes directly to the fallopian tube (zygote intrafallopian transfer--ZIFT) has been performed in women with intact oviducts. However, these attempts only slightly increased the fertility and birth rates after IVF (Edwards R G [1995] Clinical approaches to increasing uterine receptivity during human implantation. Hum Reprod 10, Suppl 3:60-67).
The effect of uterine environment on fertility rates after IVF may be equally important. It has been well established that the successful establishment of pregnancy after embryo transfer requires both a healthy blastocyct and a receptive uterus. Embryo transferred to an inadequately primed uterus are unlikely to implant. In all mammals, the endometrium, is receptive for implantation only during the specific period of time after ovulation. This phase of the luteal phase is called "implantation window". In women, the successful implantation may only take place between days 15-20 of a histologically defined 28-day cycle, i.e. during the period of highest progesterone levels (Navot D, Scott R T, Droesch K D, Veeck L L, Hung-Ching Liu, Rosenvaks Z [1991], The window of embryo transfer and efficiency of human conception in vitro. Fertil Steril 55:114-118). The optimum condition for implantation was estimated on days 20-22 of the normal cycle, i.e. 7 days after the LH surge (Bergh P A, Navot D [1992], The impact of embryonic development and endometrial maturity on the timing of implantation. Fertil Steril 58:537-542).
Adequate progesterone priming of the endometrium is essential for a successful implantation, and treatment with an antiprogestin during the luteal phase will completely prevent implantation (Chwalisz K, Stockemann K, Fuhrmann U, Fritzemeier K H, Einspanier A, Garfield R E [1995] Mechanism of action of antiprogestins in the pregnant uterus. In Henderson D, Philibert D, Roy A K, Teutsch G (eds) Steroid Receptors and Antihormones. Ann N. Y. Acad Sci 761:202-224). In the fertile cycle, progesterone regulates the transport of the fertilized egg through the oviduct and induces secretory changes required for implantation in the endometrium. Implantation is a precisely timed event in mammals. The secretory endometrial proteins (Beier H M, Elger W, Hegele-Hartung C, Mootz U, Beier-Hellwig K [1992] Dissociation of corpus luteum, endometrium and blastocyst in human implantation research. J Reprod Fert 92:511-523), and probably other intracellular and cell surface proteins, such as integrins, cytokines and growth factors produced by endometrial epithelial cells as a result of progesterone stimulation, are necessary for implantation to take place (Edwards R G [1995] Physiological and molecular aspects of human implantation. Hum Reprod 10, Suppl 2:1-14).
The asynchrony between embryo and endometrial development has been previously recognized as one of possible causes of implantation failures after IVF (Beier H M, Elger W, Hegele-Hartung C, Mootz U, Beier-Hellwig K [1992] Dissociation of corpus luteum, endometrium and blastocyst in human implantation research. J Reprod Fert 92:511-523). However, no effective methods to increase the implantation rates are available to date. The most advanced stages of human implantation are chracterized by the invasion of trophoblastic cells into the decidua and angiogenesis (Loke Y W, King A [1995] Human Implantation. Cell biology and immunology. Cambridge University Press). This stages are also dependent on progesterone, since progesterone antagonists also disrupt early pregnancy (Chwalisz K, Stockemann K, Fuhrmann U, Fritzemeier K H, Einspanier A, Garfield R E [1995] Mechanism of action of antiprogestins in the pregnant uterus. In Henderson D, Philibert D, Roy A K, Teutsch G (eds) Steroid Receptors and Antihormones. Ann N. Y. Acad Sci 761:202-224). During early pregnancy, an adequate blood flow to the uterus is essential for embryo development. An impaired blood flow to the uterus can jeopardize the establishment of pregnancy (Edwards R G (1995) Clinical approaches to increasing uterine receptivity during human implantation. Hum Reprod 10, Suppl 3:60-67). Patients with an impeded blood flow have been given aspirin to improve their blood flow. Low dose aspirin is thought to increase the prostacyclin to thromboxane A2 ratio and thereby to increase placental perfusion. However; the aspirin effect on uterine blood flow were only marginal (Goswamy R K, Williams G, Steptoe P C [1988], Decreased uterine perfusion- a cause of infertility. Hum. Reprod 3955-959; Wada I, Hsu C C; Williams G, Macnamee M C, Brinsden P R [1994], The benefits of low-dose-aspirin therapy in women with impaired uterine perfusion during assisted conception. Hum Reprod 9:1954-1957).
One of the most exciting recent advances in biology and medicine is the discovery that nitric oxide is produced by endothelial cells and that its is involved in the regulation of vascular tone, platelet aggregation, neurotransmission and immune activation. Nitric oxide is an important mediator of relaxation of the muscular smooth muscle and was formerly known as EDRF (endothelin-derived relaxing factor) (Furchgott R F and Zawadzki J V [1980], The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:373-376; Moncada S, Palmer R M G and Higgs E A [1991], Nitric oxide; physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109-14. Nitric oxide is synthesized by the oxidative deamination of a guanidino nitrogen of L-arginine by at least three different isoforms of a flavin-containing enzyme, nitric oxide synthase (Moncada S, Palmer R M G and Higgs E A [1991], Nitric oxide; physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109-144. Synthesis of nitric oxide has been shown to be competitively inhibited by analogues of L-arginine; NG-nitro-L-arginine methyl ester (L-NAME), NG-monoethyl-L-arginine (LMMA), N-iminoethyl-L-arnithine (L-NIO), L-monomethyl-L-arginine (L-NNMA) and L-NG-methylarginine (LNMA) and Nw-nitro-L-arginine (L-NA).
Nitric oxide elevates levels of cGMP (1,4,5-cyclic guanosine monophosphate) within the vascular smooth muscle to produce relaxation and to reduce blood vessels tone (Moncada S, Palmer R M G and Higgs E A. [1991], Nitric oxide; physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109-142). Nitric oxide binds to heme and thus activates soluble guanylate cyclase (Ignarro L J [1991], Physiological significance of Nitric oxide. Seminars in Perinatology 15:20-26) to increase the cellular content of cGMP. It has long been recognized that nitrovasodilators, such as sodium nitroprusside and nitroglycerin, inhibit vascular smooth muscle contractility to produce relaxation or to reduce vascular tone. These agents have been used since the late 1980s as vasodilators. However, only recently has the mechanism of action of these compounds been realized. Nitrovasodilators are now classified as nitric oxide donors because they are metabolized or spontaneously release nitric oxide (Moncada S, Palmer R M G and Higgs E A. [1991], Nitric oxide; physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109-142). The long-used nitrovasodilators may be regarded as substitution therapy for a failing physiological mechanism. Nitric oxide is also produced by macrophages and other immune cells.
Three highly related NOS enzymes have been isolated and identified. These Include endothelial NOS (e-NOS, type III), neuronal NOS (n-NOS, type II) and inducible NOS (i-NOS, type I) (Knowles R G and Moncada S [1994], Nitric oxide synthases in mammals. Biochem J 298:249-258; Sessa W C. [1994], The Nitric Oxide Synthase Family of Proteins. J Vasc Res 1994; 31:131-143; Nathan C [1992], Nitric oxide as a secretory product of mammalian cells. FASEB J 6:301-3064). The constitutive isoforms e-NOS and b-NOS were originally identified in endothelial and neuronal tissues, respectively, and they rapidly and transiently produce small amounts of NO under basal conditions. The i-NOS isoform is inducible by cytokines or endotoxin (LPS) and it produces large quantities of NO for hours or days in a Ca.sup.2+ -independent manner. Cells expressing iNOS do not generate NO under basal conditions. The e-NOS form of the enzyme is expressed in endothelial cells, in cardiac myocytes, platelets and some neurones. The e-NOS-derived NO is the most important vasodilator. It is released In low levels to maintain a constant vasorelaxation and maintain normal blood pressure. The n-NOS isoform is thought to act as a neurotransmitter. It is thought to be important in mediating such functions as gastrointestinal motility and penile erection.
There is a substantial body of evidence from animal experiments that a deficiency in nitric oxide contributes to the pathogenesis of a number of diseases, including hypertension, atherosclerosis and diabetes (Moncada S, Palmer R M G and Higgs E A [1991], Nitric oxide; physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109-142). There are many recent studies showing that the inhibition of nitric oxide synthase dramatically increases blood pressure. Treatment of pregnant rats and guinea pigs with nitric oxide synthase inhibitors produce symptoms identical to preeclampsia (Chwalisz K and Garfield R E [1994], Role of progesterone during pregnancy: Models of parturition and preeclampsia. Z. Geburtsh. u. Perinat. 198:170-180). Preeclampsia is characterized by increased blood pressure and peripheral vascular resistance, fetal growth retardation, proteinuria and edema. In humans, histopathologic and clinical (fetal growth retardation, fetal death) evidence indicate that reduced placental perfusion is the earliest and most consistent change observed in preeclampsia (Roberts J M and Redman C W G. [1993], Pre-eclampsia: more than pregnancy-induced hypertension 341:1447-1451; Friedman E A 1988], Preeclampsia: a review of the role of prostaglandins. Obstet Gynecol 71:122-137).
The L-arginine-NO system is present in the uterus (Garfield R E and Yallampalli C. [1993] Control of myometrial contractility and labor. In: Basic Mechanisms Controlling Term and Preterm Birth. ed: K Chwalisz, R E Garfield, Springer-Verlag, New York, pp. 1-29; Chwalisz K and Garfield R E. [1994], Antiprogestins in the Induction of labor. Ann New York Acad Scie 734:387-413; Buhimschi I, Yallampalli C, Dong Y-L and Garfield R E. [1995], Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy. Am J Obstet Gynecol 172:1577-1584; Sladek S M, Regenstrin A C, Lykins D. et al. [1993], Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy. Am J Obstet Gynecol 169:1285-1291). This system plays an important role in control of uterine contractility, pregnancy maintenance, onset of labor and also fetal perfusion. L-arginine and nitric acid caused a rapid and substantial relaxation of spontaneous activity of the uterine strips from rats at mid to near term gestation (Buhimschi I, Yallampalli C, Dong Y-L and Garfield R E [1995] Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy. Am J Obstet Gynecol 172:1577-1584; Garfield R E and Yallampalli C. [1993] Control of myometrial contractility and labor. In: Basic Mechanisms Controlling Term and Preterm Birth. ed: K. Chwalisz, R E Garfield, Springer-Verlag, New York, pp. 1-29; Sladek S M, Regenstrin A C, Lykins D. et al. [1993] Nitric oxide synthase activity in pregnant rabbit uterus decreases on the last day of pregnancy. Am J Obstet Gynecol 169:1285-1291; Natuzzi E S, Ursell P C, Harrison M. et al [1993], Nitric oxide synthase activity in the pregnant uterus decreases at parturition. Biochem Biophys Res Commun 194:108-114; Jennings R W, MacGillvray T E and Harrison M R. [1995], Nitric oxide inhibits preterm labor in the rhesus monkey. J Mat Fet Med 2:170-175).
The expression of NOS enzymes in the rat uterus was studied with immunoblotting with monoclonal antibodies, i-NOS and e-NOS were detected in the uterus (myometrium) The uterine i-NOS enzyme decreased in the uterus during labor at term and preterm in animals treated to deliver prematurely. Opposite changes were observed in the cervix (Buhimschi I, Ali M, Jain V, Chwalisz K and Garfield R E. [1996], Differential regulation of nitric oxide in the uterus and cervix during pregnancy and labor. Human Reproduction [in press]).
NOS is also present in placental tissues and uterine arteries. The trophoblast invasion of uteroplacental arteries in relation to the NO synthase isoform expression was studied in pregnant guinea pigs by means of immune- and histochemistry as compared to arterial dilatation. A pronounced dilatation of uteroplacental arteries begins at mid-pregnancy and progresses until term (Nanaev A, Chwalisz K, Frank H-G, Kohnen G, Hartung C-H and Kaufmann P. [1995], Physiological dilation of uteroplacental arteries in the guinea pig depends upon nitric oxide synthase activity of extravillous prophoblast. Cell Tissue Res:282:407-421). This study demonstrates that dilatation of uteroplacental arteries can be seen when Invading trophoblast cells coexpressing endothelial (e-NOS) and macrophage (iNOS) nitric oxide synthase are found in the vicinity of the vessels, i.e. prior to trophoblast invasion of the arterial walls. Conrad et al.,(1993), localized NOS to the syncythiotrophoblast cell layer in human placenta (Conrad K P, Vill M, Mcguire P G, Dail W G, Davis A K [1993], Expression of nitric oxide synthase by syncythiotrophoblast in human placental villi, FASEB J 7:1269-1276). Morris et al., (1993), demonstrated both calcium-dependent and calcium-independent activity in human placental villi and the basal plate (Morris N H, Sooranna S R, Eaton B M, Steer P J (1993) NO synthase activity in placental bed and tissues from normotensive pregnant women. Lancet 342:679-680), and Myatt et al (1993), showed that placental villous tree synthesized a calcium-dependent-isoform of the NOS (Myatt L, Brockman D E, Langdon G, Pollock J S [1993], Constitutive calcium-dependent isoform of nitric oxide synthase in the human placenta villous vascular tree. Placenta 14:373-383; Myatt L, Brockman D E, Eis A L, Pollock J S [1993] Immunohistochemical Iolalization of nitric oxide synthase in the human placenta. Placenta 14:487-495). In addition, Buttery et al., (1994) showed that endothelial NOS at term was localized in the endothelium of umbilical artery and vein and in the placental syncythiotrophoblast (Buttery L D K, McCarthy A, Springall A et al., [1994], Endothelial nitric oxide synthase in the human placenta: regional distribution and proposed regulatory role at feto-maternal interface. Placenta 15:257-267). Furthermore, Moorhead et al., (1995) have shown that NADPH diaphorase (non-specific reaction to identify nitric oxide synthase) was in various uterine components during early pregnancy (Moorhead C S, Lawhun M, Nieder G L [1995], Localization of NADPH diaphorase in the mouse uterus during the first half of pregnancy and during an artificially-induced decidual cell reaction. J Histochem Cytochem 43:1053-1060). Finally, Toth et al., (1995) demonstrated that NOS activity was present in the first trimester human placental homogenates (Toth M, Kukor Z, Romero R,. Hertelendy F [1995], Nitric oxide synthase in first trimester human placenta: Characterization and subcellular distribution. Hypertens Pregnancy 14/3:287-300).
These studies suggest that nitric oxide is an important factor regulating placental blood flow and myometrial quiescence during pregnancy. However, there are no studies published to date which demonstrate the detrimental effects of NOS inhibition on implantation or the beneficial effects of nitric oxide donors or substrates on implantation after IVF or in women with early pregnancy loss. In contrast, Haddad et al., (1995) suggested that the increased nitric oxide production is associated with early embryo loss in mice and that iNOS inhibitors can by used to treat early abortion (Haddad E K, Duclos A J, Baines M G [1995], Early embryo loss is associated with local production of nitric oxide by decidual mononuclear cells. J Exp Med 182:1143-51).